In this study neutral and acidic oligosaccharide fractions prepared from human milk have been investigated using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The fraction of neutral oligosaccharides was separated by gel permeation chromatography (GPC) and the resulting subfractions were analyzed by MALDI-MS using the positive ion mode. Several low-molecular-weight glycans (degree of polymerization up to 13) were observed whose structures have already been elucidated. In addition, a variety of so far unknown large-sized carbohydrates was detected whose molecular weights range from M(r) 2242 to 8000. The large-sized glycans which possess a low abundance appear to be composed of both lactosamine and fucose residues attached to the lactose unit at the reducing end of the sugar chains with a highly variable stochiometry. Following subfractionation by GPC, acidic (i.e., containing sialic acid) glycans were analyzed by MALDI-MS using both positive and negative ion mode. Because of the inferior stability of acidic glycans, various matrices were applied and compared with respect to signal intensity, resolution, and analyte stability.